Stiffening and unfolding of early deposited-fibronectin increase proangiogenic factor secretion by breast cancer-associated stromal cells.

Fibronectin (Fn) forms a fibrillar network that controls cell behavior in both physiological and diseased conditions including cancer. Indeed, breast cancer-associated stromal cells not only increase the quantity of deposited Fn but also modify its conformation. However, (i) the interplay between mechanical and conformational properties of early tumor-associated Fn networks and (ii) its effect on tumor vascularization remain unclear. Here, we first used the Surface Forces Apparatus to reveal that 3T3-L1 preadipocytes exposed to tumor-secreted factors generate a stiffer Fn matrix relative to control cells. We then show that this early matrix stiffening correlates with increased molecular unfolding in Fn fibers, as determined by Förster Resonance Energy Transfer. Finally, we assessed the resulting changes in adhesion and proangiogenic factor (VEGF) secretion of newly seeded 3T3-L1s, and we examined altered integrin specificity as a potential mechanism of modified cell-matrix interactions through integrin blockers. Our data indicate that tumor-conditioned Fn decreases adhesion while enhancing VEGF secretion by preadipocytes, and that an integrin switch is responsible for such changes. Collectively, our findings suggest that simultaneous stiffening and unfolding of initially deposited tumor-conditioned Fn alters both adhesion and proangiogenic behavior of surrounding stromal cells, likely promoting vascularization and growth of the breast tumor. This work enhances our knowledge of cell - Fn matrix interactions that may be exploited for other biomaterials-based applications, including advanced tissue engineering approaches

Engineering mechanobiology: the bacterial exclusively-mechanosensitive ion channel MscL as a future tool for neuronal stimulation technology

The development of novel approaches to stimulate neuronal circuits is crucial to understand the physiology of neuronal networks, and to provide new strategies to treat neurological disorders. Nowadays, chemical, electrical or optical approaches are the main exploited strategies to interrogate and dissect neuronal circuit functions. However, although all these methods have contributed to achieve important insights into neuroscience research field, they all present relevant limitations for their use in in-vivo studies or clinical applications. For example, while chemical stimulation does not require invasive surgical procedures, it is difficult to control the pharmacokinetics and the spatial selectivity of the stimulus; electrical stimulation provides high temporal bandwidth, but it has low spatial resolution and it requires implantation of electrodes; optical stimulation provides subcellular resolution but the low depth penetration in dense tissue still requires the invasive insertion of stimulating probes. Due to all these drawbacks, there is still a strong need to develop new stimulation strategies to remotely activate neuronal circuits as deep as possible. The development of remote stimulation techniques would allow the combination of functional and behavioral studies, and the design of novel and minimally invasive prosthetic approaches. Alternative approaches to circumvent surgical implantation of probes include transcranial electrical, thermal, magnetic, and ultrasound stimulation. Among v these methods, the use of magnetic and ultrasound (US) fields represents the most promising vector to remotely convey information to the brain tissue. Both magnetic and low-intensity US fields provide an efficient mean for delicate and reversible alteration of cells and tissues through the generation of local mechanical perturbations. In this regard, advances in the mechanobiology research field have led to the discovery, design and engineering of cellular transduction pathways to perform stimulation of cellular activity. Furthermore, the use of US pressure fields is attracting considerable interest due to its potential for the development of miniaturized, portable and implantation-free US stimulation devices. The purpose of my PhD research activity was the establishment of a novel neuronal stimulation paradigm adding a cellular selectivity to the US stimulation technology through the selective mechano-sensitization of neuronal cells, in analogy to the well-established optogenetic approach. In order to achieve the above mentioned goal, we propose the cellular overexpression of mechanosensitive (MS) ion channels, which could then be gated upon the application of an US generated pressure field. Therefore, we selected the bacterial large conductance mechanosensitive ion channel (MscL), an exclusively-MS ion channel, as ideal tool to develop a mechanogenetic approach. Indeed, the MscL with its extensive characterization represents a malleable nano-valve that could be further engineered with respect to channel sensitivity, conductance and gating mechanism, in order to obtain the desired biophysical properties to achieve reliable and efficient remote mechanical stimulation of neuronal activity. In the first part of the work, we report the development of an engineered MscL construct, called eMscL, to induce the heterologous expression of the bacterial protein in rodent primary neuronal cultures. Furthermore, we report the structural and functional characterization of neuronal cells expressing the eMscL channel, at both single-cell and network levels, in order to show that the functional expression of the engineered MscL channel induces an effective vi neuronal sensitization to mechanical stimulation, which does not affect the physiological development of the neuronal itself. In the second part of the work, we report the design and development of a water tank-free ultrasound delivery system integrated to a custom inverted fluorescence microscope, which allows the simultaneous US stimulation and monitoring of neuronal network activity at single resolution. Overall, this work represents the first development of a genetically mechanosensitized neuronal in-vitro model. Moreover, the developed US delivery system provides the platform to perform high-throughput and reliable investigation, testing and calibration of the stimulation protocols. In this respect, we propose, and envisage in the near future, the exploitation of the engineered MscL ion channel as a mature tool for novel neuro-technological applications

Introducing monitoring and automation in cartilage tissue engineering, toward controlled clinical translation

The clinical application of tissue engineered products requires to be tightly connected with the possibility to control the process, assess graft quality and define suitable release criteria for implantation. The aim of this work is to establish techniques to standardize and control the in vitro engineering of cartilage grafts. The work is organized in three sub-projects: first a method to predict cell proliferation capacity was studied, then an in line technique to monitor the draft during in vitro culture was developed and, finally, a culture system for the reproducible production of engineered cartilage was designed and validated. Real-time measurements of human chondrocyte heat production during in vitro proliferation. Isothermal microcalorimetry (IMC) is an on-line, non-destructive and high resolution technique. In this project we aimed to verify the possibility to apply IMC to monitor the metabolic activity of primary human articular chondrocytes (HAC) during their in vitro proliferation. Indeed, currently, many clinically available cell therapy products for the repair of cartilage lesions involve a process of in vitro cell expansion. Establishing a model system able to predict the efficiency of this lengthy, labor-intensive, and challenging to standardize step could have a great potential impact on the manufacturing process. In this study an optimized experimental set up was first established, to reproducible acquire heat flow data; then it was demonstrated that the HAC proliferation within the IMC-based model was similar to proliferation under standard culture conditions, verifying its relevance for simulating the typical cell culture application. Finally, based on the results from 12 independent donors, the possible predictive potential of this technique was assessed. Online monitoring of oxygen as a non-destructive method to quantify cells in engineered 3D tissue constructs. This project aimed at assessing a technique to monitor graft quality during production and/or at release. A quantitative method to monitor the cells number in a 3D construct, based on the on-line measurement of the oxygen consumption in a perfusion based bioreactor system was developed. Oxygen levels dissolved in the medium were monitored on line, by two chemo-optic flow-through micro-oxygen sensors connected at the inlet and the outlet of the bioreactor scaffold chamber. A destructive DNA assay served to quantify the number of cells at the end of the culture. Thus the oxygen consumption per cell could be calculated as the oxygen drop across the perfused constructs at the end of the culture period and the number of cells quantified by DNA. The method developed would allow to non-invasively monitoring in real time the number of chondrocytes on the scaffold. Bioreactor based engineering of large-scale human cartilage grafts for joint resurfacing. The aim of this project was to upscale the size of engineered human cartilage grafts. The main aim of this project consisted in the design and prototyping of a direct perfusion bioreactor system, based on fluidodynamic models (realized in collaboration with the Institute for Bioengineering of Catalonia, Spain), able to guarantee homogeneous seeding and culture conditions trough the entire scaffold surface. The system was then validated and the capability to reproducibly support the process of tissue development was tested by histological, biochemical and biomechanical assays. Within the same project the automation of the designed scaled up bioreactor system, thought as a stand alone system, was proposed. A prototype was realized in collaboration with Applikon Biotechnology BV, The Netherlands. The developed system allows to achieve within a closed environment both cell seeding and culture, controlling some important environmental parameters (e.g. temperature, CO2 and O2 tension), coordinating the medium flow and tracking culture development. The system represents a relevant step toward process automation in tissue engineering and, as previously discussed, enhancing the automation level is an important requirement in order to move towards standardized protocols of graft generation for the clinical practice. These techniques will be critical towards a controlled and standardized procedure for clinical implementation of tissue engineering products and will provide the basis for controlled in vitro studies on cartilage development. Indeed the resulting methods have already been integrated in a streamlined, controlled, bioreactor based protocol for the in vitro production of up scaled engineered cartilage drafts. Moreover the techniques described will serve as the foundation for a recently approved Collaborative Project funded by the European Union, having the goal to produce cartilage tissue grafts. In order to reach this goal the research based technologies and processes described in this dissertation will be adapted for GMP compliance and conformance to regulatory guidelines for the production of engineered tissues for clinical use, which will be tested in a clinical trial

Surface and bulk stresses drive morphological changes in fibrous microtissues

Engineered fibrous tissues consisting of cells encapsulated within collagen gels are widely used three-dimensional in vitro models of morphogenesis and wound healing. Although cell-mediated matrix remodeling that occurs within these scaffolds has been extensively studied, less is known about the mesoscale physical principles governing the dynamics of tissue shape. Here, we show both experimentally and by using computer simulations how surface contraction through the development of surface stresses (analogous to surface tension in fluids) coordinates with bulk contraction to drive shape evolution in constrained three-dimensional microtissues. We used microelectromechanical systems technology to generate arrays of fibrous microtissues and robot-assisted microsurgery to perform local incisions and implantation. We introduce a technique based on phototoxic activation of a small molecule to selectively kill cells in a spatially controlled manner. The model simulations, which reproduced the experimentally observed shape changes after surgical and photochemical operations, indicate that fitting of only bulk and surface contractile moduli is sufficient for the prediction of the equilibrium shape of the microtissues. The computational and experimental methods we have developed provide a general framework to study and predict the morphogenic states of contractile fibrous tissues under external loading at multiple length scales.Published versio

Focus on the Physics of Cancer

Despite the spectacular achievements of molecular biology in the second half of the twentieth century and the crucial advances it permitted in cancer research, the fight against cancer has brought some disillusions. It is nowadays more and more apparent that getting a global picture of the very diverse and interlinked aspects of cancer development necessitates, in synergy with these achievements, other perspectives and investigating tools. In this undertaking, multidisciplinary approaches that include quantitative sciences in general and physics in particular play a crucial role. This `focus on' collection contains 19 articles representative of the diversity and state-of-the-art of the contributions that physics can bring to the field of cancer research.Comment: Invited editorial review for the `Focus on the Physics of Cancer' published by the New journal of Physics in 2011--201

Larval condition and growth of Sardinella brasiliensis (Steindachner, 1879): preliminary results from laboratory studies

Brazilian sardine, the most important resource along the southeastern Brazilian coast, presented great variations and declines in its stocks. The main factors contributing to this are: oceanographic structure changes; recruitment failures; excessive catches of juveniles and increase in fishery effort. In spite of this, no alterations in the density-dependent parameters were detected. Consequently, methods analysing the condition of the larvae coupled with methods determining growth using sagittae otolith increment width were applied to evaluate growth under experimental conditions. The results of the readings on the sagittae were compared with the age of the laboratory-reared sardine larvae and confirmed that increments are formed on a daily basis. Under poor feeding conditions, sardine larvae showed a low growth expressed by dry weight, RNA/DNA ratio and tryptic enzyme activity and by the narrow and low contrast increments in the otoliths. The results of the biochemical indices showed an unexpected decline in the feeding group coupled with a decrease in width of increment numbers 8 and 10. Other factors than food availability were affecting the condition of the larvae and might be indicative of physiological processes and ontogenetic changes occurring in sardine larvae

Modular Instrumentation for Controlling and Monitoring In-Vitro Cultivation Environment and Image-based Functionality Measurements of Human Stem Cells

Artificial animal cell culture was successfully developed by Ross Harrison in 1907. But it was not until the 1940’s and 1950’s that several developments occurred, which expedited the cell culturing in-vitro (C-Vitro) to be a consistent and reproducible technique to study isolated living-cells in a controlled environment. Currently, CVitro is one of the major tools in cellular and molecular biology both in the academia and industry. They are extensively utilised to study the cellular physiology/biochemistry, to screen drugs/therapeutic compounds, to understand the effects of drugs/toxic compounds and also to identify the pathways of carcinogenesis/mutagenesis. It is also used in large scale manufacturing of vaccines and therapeutic proteins. In any experimental setup, it is important that the C-Vitro model should represent the physiological phenomena of interest with reasonable accuracy so that all experimental results are statistically consistent and reproducible. In this direction, sensors and measurement systems play important roles in in-situ detection and/or control/manipulation of cells/tissues/environment. This thesis aimed to develop new technology for tailored cell culturing and integrated measurements. Firstly, design and assembly of a portable Invert-upright microscope interchangeable modular cell culturing platform (iuCMP) was envisioned. In contrast to conventional methods, micro-scaled systems mimic the cells' natural microenvironment more precisely, facilitating accurate and tractable models. The iuCMP integrates modular measurement schemes with a mini culture chamber using biocompatible cell-friendly materials, automated environment-control (temperature and gas concentrations), oxygen sensing and simultaneous functional measurements (electrophysiological and image-based). Time lapse microscopy is very useful in cell biology, but integration of advanced >i>in-vitro/device based biological systems (e.g. lab/organ/body-on-chips, or mini-bioreactors/microfluidic systems) into conventional microscopes can be challenging in several circumstances due to multiple reasons. But in iuCMP the main advantage is, the microscope can be switched either as an inverted or as an upright system and therefore can accommodate virtually any in-vitro device. It can capture images from regions that are otherwise inaccessible by conventional microscopes, for example, cells cultured on physical or biochemical sensor systems. The modular design also allows accommodating more sensor or measurement systems quite freely. We have demonstrated the system for video-based beating analysis of cardiomyocytes, cell orientation analysis on nanocellulose, and simultaneous long-term in-situ microscopy with oxygen and temperature sensing in hypoxia. In an example application, the system was utilised for long-term temperature stressing and simultaneous mechanobiological analysis of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). For this the iuCMP together with a temperature sensor plate (TSP) and a novel non-invasive beating analysis software (CMaN—cardiomyocyte function analysis tool, scripted as a subpart of this thesis), was applied for automated temperature response studies in hiPSC-CM cultures. In-situ temperature sensing is usually challenging with bulky external sensors, but TSPs solved this issue. In the temperature response study, we showed that the relationship between hiPSC-CM beating frequency and temperature is non-linear and measured the Q10 temperature coefficients. Moreover, we observed the hiPSC-CM contractile networking, including propagation of the action potential signal between dissociated clusters and their non-invasive measurements. It was the first case where these events were reported in hiPSC-CM clusters and their noninvasive measurements by image processing. The software CMaN comes with a user-friendly interface and, is equipped with features for batch processing, movement centre detection and cluster finding. It can extract six different signals of the contractile motion of cardiomyocytes (clusters or single cells) per processing. This ensures a minimum of one useful beating signal even in the cases of complex beating videos. On the processing end, compared to similar tools, CMaN is faster, more sensitive, and computationally less expensive and allows ROI based processing. In the case of healthy cells, the waveform of the signal from the CMaN resembles an ECG signal with positive and negative segments, allowing the computation of contraction and relaxation features separately. In addition to iuCMP, a Modular optical pH measurement system (MO-pH) for 24/7 non-contact cell culture measurements was also developed. The MO-pH incorporates modular sterilisable optical parts and is used in phenol-red medium cell cultures. The modular assembly of MO-pH cassettes is unique and reusable. Measurements are carried out in a closed flow system without wasting any culture medium and requires no special manual attention or recalibrations during culture. Furthermore, a new absorption correction model was put forward that minimised errors caused e.g. by biolayers in spectrometric pH measurement, which improved the pH measurement accuracy. MO-pH has been applied in long-term human adipose stem cells (hASC) expansion cultures in CO2 dependent and independent media. Additionally, the MO-pH was also utilised to comprehend the behaviour of pH, temperature and humidity in water jacked incubators as well as to record the pH response as a function of temperature in the presence and absence of CO2 in the context of stem cell cultures. The resulting plots clearly showed the interplay between measured parameters indicating a few stress sources present all through the culture. Additionally, it provided an overall picture of behaviour of critical control parameters in an incubator and pointed out the need for bioprocess systems with automatic process monitoring and smart control for maximum yield, optimal growth and maintenance of the cells. Besides, we also integrated MO-pH into flasks with reclosable lids (RL-F) and tested its applicability in stem cell cultures. A standalone system around an RL-F flask was built by combining the cell culture, medium perfusion and optical measurements. The developed RL-F system has been successfully tested in ASC-differentiation cultures. Finally, a few trial experiments for image-based pH estimation aimed for iuCMP have also been carried out. This includes tests with LCD illumination, optical projection tomography, and webcam systems. In reality, the pH is not distributed uniformly in tissues, and has shown a gradient of up to 1.0 pH unit within 1 cm distance. Therefore, producing reliable pH maps also in in-vitro can be important in understanding various common pathologies and location of lesions. A reliable and adequately developed long-term pH mapping method will be an important addition into the iuCMP

Aquaporin 5 Interacts with Fluoride and Possibly Protects Against Caries

Aquaporins (AQP) are water channel proteins and the genes coding for AQP2, AQP5, and AQP6 are clustered in 12q13. Since AQP5 is expressed in serous acinar cells of salivary glands, we investigated its involvement in caries. DNA samples from 1,383 individuals from six groups were studied. Genotypes of eight single nucleotide polymorphisms covering the aquaporin locus were tested for association with caries experience. Interaction with genes involved in enamel formation was tested. The association between enamel microhardness at baseline, after creation of artificial caries lesion, and after exposure to fluoride and the genetic markers in AQP5 was tested. Finally, AQP5 expression in human whole saliva, after exposure to fluoride in a mammary gland cell line, which is known to express AQP5, and in Wistar rats was also verified. Nominal associations were found between caries experience and markers in the AQP5 locus. Since these associations suggested that AQP5 may be inhibited by levels of fluoride in the drinking water that cause fluorosis, we showed that fluoride levels above optimal levels change AQP5 expression in humans, cell lines, and rats. We have shown that AQP5 is involved in the pathogenesis of caries and likely interact with fluoride.Fil: Anjomshoaa, Ida. University of Pittsburgh; Estados UnidosFil: Briseño Ruiz, Jessica. University of Pittsburgh; Estados UnidosFil: Deeley, Kathleen. University of Pittsburgh; Estados UnidosFil: Poletta, Fernando Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. CEMIC-CONICET. Centro de Educaciones Médicas e Investigaciones Clínicas "Norberto Quirno". CEMIC-CONICET.; ArgentinaFil: Mereb, Juan C.. Provincia de Río Negro. Ministerio de Salud. Hospital de Área El Bolsón ; ArgentinaFil: Leite, Aline L.. Universidade de Sao Paulo; BrasilFil: Barreta, Priscila A. T.. Universidade de Sao Paulo; BrasilFil: Silva, Thelma L.. Universidade de Sao Paulo; BrasilFil: Dizak, Piper. University of Pittsburgh; Estados UnidosFil: Ruff, Timothy. University of Pittsburgh; Estados UnidosFil: Patir, Asli. İstanbul Medipol Üniversitesi; TurquíaFil: Koruyucu, Mine. İstanbul Üniversitesi; TurquíaFil: Abbasoğlu, Zerrin. Yeditepe Üniversitesi; TurquíaFil: Casado, Priscila L.. Universidade Federal Fluminense; BrasilFil: Brown, Andrew. University of Pittsburgh; Estados UnidosFil: Zaky, Samer H.. University of Pittsburgh; Estados UnidosFil: Bayram, Merve. İstanbul Medipol Üniversitesi; TurquíaFil: Küchler, Erika C.. University of Pittsburgh; Estados UnidosFil: Cooper, Margaret E.. University of Pittsburgh; Estados UnidosFil: Liu, Kai. University of Pittsburgh; Estados UnidosFil: Marazita, Mary L.. University of Pittsburgh; Estados UnidosFil: Tanboğa, İlknur. Marmara Üniversitesi; TurquíaFil: Granjeiro, José M.. Universidade Federal Fluminense; Brasil. Instituto Nacional de Metrologia, Qualidade e Tecnologia; BrasilFil: Seymen, Figen. İstanbul Üniversitesi; TurquíaFil: Castilla, Eduardo Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. CEMIC-CONICET. Centro de Educaciones Médicas e Investigaciones Clínicas "Norberto Quirno". CEMIC-CONICET.; Argentina. Fundación Oswaldo Cruz; BrasilFil: Orioli, Iêda M.. Universidade Federal do Rio de Janeiro; BrasilFil: Sfeir, Charles. University of Pittsburgh; Estados UnidosFil: Owyang, Hongjiao. Marmara Üniversitesi; TurquíaFil: Rabelo Buzalaf, Marilia Afonso. Universidade de Sao Paulo; BrasilFil: Vieira, Alexandre R.. University of Pittsburgh; Estados Unido

Toward disease-specific therapies in mind-body cancer research: reverse engineering, epigenetic feedback and in vitro/ in vivo combination protocols

Although we have significant experimental evidence demonstrating that specific meditation forms correlate with particular effects on biological targets, mind-body  therapeutic applications are still very rudimentary and poorly standardized, consisting of little more than exercises designed to trigger a parasympathetic response.  If the sole physiological effect of meditation were related to the relaxation response, then indeed most forms of meditation would be expected to work in similar ways and achieve similar results. But as we have explored in a previous panel discussion anchored by Michael Persinger and his group [Bajpai et al., Journal of Nonlocality, II-2, 2013], the convergence of photobiology  and qigong experimental research  indicates that specific brainwave patterns correlate with specific biophoton emission frequencies, microtubule conformational states  and biological effects, both at the level of the operator’s body and in remote targets. Of greatest clinical  interest is the ability of focused intent to produce target-specific, directional effects, while leaving control samples unaffected – a feature that has been documented by over a hundred  in vitro and in vivo controlled qigong experiments and corroborated by several hundred  Random Event Generator (REG) studies conducted at Princeton and other university labs.  While the physical modeling of such remote effects is still speculative, the potential applications are sufficiently intriguing to warrant an empirical  leap ahead of the theoretical staging.  If cancer is “a disease of geometry” due to a  “misregulation of the field of information that orchestrates individual cells’ activities towards normal anatomy”, as Chernet and Levin argue [Chernet and Levin, J Clin Exp Oncol 2013, S1], could we find a way to design  and calibrate specific meditation forms to predictably achieve intended electromagnetic effects  at a biological target (such as  a tumor)?  The present paper proposes a general approach that might  take us a step closer to tailoring such targeted mind-body interventions  through the use of reverse engineering, rapid-expression epigenetic feedback and an in vitro/ in vivo combination protocol

Near infrared laser irradiation on single multicellular spheroids

Light is being widely used in biomedicine due to its non-invasive nature, with application in imaging techniques and as a therapeutic agent. However, several aspects of its effect on irradiated tissues still leads to discussion in the scientific community. This particularly relates to novel biological models, as is the case of 3D multicellular spheroids, which are rising as an intermediate model between in vitro monolayer cultures and small animals. The applications of these spherical cell aggregates are diverse and include tissue reconstruction, drug testing or cancer studies, to cite some. To address the effect of light on these models, we use spheroids formed by MCF-7 (adenocarcinoma) or by U-87 MG (glioblastoma) cells. After their growth, they have been irradiated individually with focused laser radiation in the near-infrared (808 nm and 1450 nm), which provokes size changes in the spheroid. Time-lapse imaging in a brightfield microscope allows to define a reduction parameter, which informs about the extent of the size change. This parameter is correlated with cell viability studies; thus, we can set a safe range of reduction in which spheroids are not damaged by irradiation, and a threshold that should be avoided to keep cell mortality low. This correlation can be used as preliminary and visual information on the survival of cells during optical experiments with 3D spheroidsBesides, we would like to mention our funding institution, Ministerio de Ciencia e Innovacion ´ de Espana ˜ (grants PID2019-105195RA-I00, PID2019-110632RB-I00, CNS2022-135495, CNS2022-135965 and TED2021-129937B-I00). P.C. thanks the regional government of Comunidad de Madrid for the Programa Investigo (Plan de Recuperacion, ´ Transformacion ´ y Resiliencia) which was developed thanks to SEPE, Ministerio de Trabajo y Economía Social and the European Union through NextGenerationE